Objective

Flowering plants are the most successful plant group on Earth and provide the basis for most human and animal food. The development of flowers is dependent on a gene regulatory network orchestrated by LEAFY (LFY), the master transcription factor. In Arabidopsis as in other flowering plants, LFY activity is dependent upon its interaction with UFO, a member of the Skp1–Cullin–F-box E3 ubiquitin ligase complex. The goal of this project is to understand why LFY requires interacting with UFO to build flowers. For this, we will apply state-of-the-art technologies including mass-spectrometry, crystallography, biochemistry and plant developmental genetics to identify and characterize the function of Post-Translational Modifications (PTM) on LFY or LFY interacting proteins, with special emphasis on ubiquitination. After identifying key residues involved in the LFY-UFO interaction or targeted by PTM, their functional importance will be assayed in planta. This project should facilitate better understanding of the mechanism of LFY activation. LEAFY and the development of flower will be used here as a model to understand the “activation by degradation” mechanism, still elusive in most organisms and poorly studied in plants. Moreover, understanding the principles governing LFY activation should provide means to modify at will the architecture of plant inflorescences. This project builds on the current background of the applicant in the field of Mass-Spectrometry combined with the outstanding expertise at Grenoble and high-throughput facilities in Structural biology and plant developmental genetics. Notably, the applicant will work within one of the top French Research Centers and in a multidisciplinary environment that will allow the fellow to acquire complementary ‘new knowledge and skills’ in structural biology and developmental genetics. This will re-enforce the fellow’s background and professional maturity for employment opportunities in both academia and industry sectors.

Importance for society: Provide insights on the mechanism of LFY activation by UFO, a key step in the making of flowers and hence the continuation of life in flowering plants. Project sheds light on the molecular mechanism of flower formation setting the means to modify at will the architecture of plant inflorescences. The outputs have application in agriculture and potentially impacting social needs on food security.

Overall Objectives:WP1. To identify LFY post-translational modifications (PTMs)WP2. To validate ubiquitinated LFY lysine residues and characterize their functional importanceWP3. To map LFY-UFO interacting domains and the role of PTMsWP4. To determine LFY interactome in planta

Month 1-12WP1 and 4. To isolate LFY and identify PTMsImmunoprecipitation (IP) of LFY in planta was challenging due to antibody leakage increasing non-specificity.

WP3. To determine the structure of LFY-UFO complexTask 3.1: Isolating and purifying LFY and UFO proteinsHis tagged LFY∆40 (lminus 40 amino acids) and truncated MBP-tagged UFO were purified from bacterial expression system aiming to isolate soluble and non-aggregating proteins (Figure 1).Figure 1. Expressed UFO constructs (A) Five UFO constructs and (B) representative image showing isolation of UFO construct recombinantly expressed in bacterial cells.Task 3.2: Determining interacting domains of UFO and LFY using LC-MS/MS (a) Co-purification: Co-purifications of MBP-UFO and HIS-LFY showed that the two could interact (WB; Figure 2).Figure 2. Western blots to visualize MBP-UFO (CM04) and HIS-LFY following co-purification. (A) panel for anti-MBP (B) anti-LFY. M- marker; FT-flow-through; W-wash; E-elution.(b) Yeast-2 hybrid assay: Various LFY constructs were used to test which region of LFY can interact with UFO. We show that UFO could interact with the C-term of LFY (Figure 3).Figure 3. Yeast-2 hybrid assays. Panel 1 shows truncations of LFY and panel 2 yeast colonies visualized after 6 days of incubation.(c) Chemical crosslinking and Mass spectrometry (MS)An MS cleavable cross-linker, disuccinimidyl sulfoxide was used to map the interacting domains between LFY and UFO and analyzed by MS. We identified 6 peptides, three from LFY and three from UFO (Table 1, Figure 4). Using LFY and UFO structural models the identified peptides are well exposed for interaction.Table 1. Peptides of UFO and LFY obtained after chemical crosslinking and MS.Figure 4. Pictorial view showing the location of the peptides identified in Table 1. (A) Peptides of LFY, (B) peptides of UFO and (C) predicted structural model of UFO indicating the peptide loops on UFO that crosslinked with LFY.Task 3.3: Determine the crystal structure of LFY-UFOThis ambitious task could not be performed due to aggregation of UFO.

Month 13-18WP1 and 4. To isolate and identify LFY interactome and PTMsLFY IP from in planta was successful using dynabeads conjugated with antiLFY antibody (Figure 5A). PTMs were assessed by WB (Figure 5B and C).Figure 5. WB depicting LFY and PTMs. IP of LFY from in planta samples using antiLFY antibody conjugated dynabeads. WB were performed against (A) antiLFY, (B) anti-Ser/Thr and antiUbiSite.In vitro phosphorylation: In collaborated with Dr. Claude Cochet, in vitro LFY phosphorylation using casein kinase (CK)II was done and CKII could phosphorylate LFY (Figure 6). LFY phosphopeptides were identified by MS. Figure 6. In vitro casein kinase II assay (A) with radiolabeled ATP. (B) different reactions of ATP with alpha subunit, alpha+LFY, and alpha-beta+LFY. (C) shows the samples in the gel after coomassie staining.

Month 19-24WP2. To characterize ubiquitinated lysinesTasks could not be executed since ubiquitinated residues were not identified by MS. However, in vitro ubiquitination assay will be performed in the future.WP3. To determine the structure of LFY-UFO complexSynthesized LFY and UFO peptides (Figure 4) were used in fluorescent anisotropy. LFY peptides showed no reaction with UFO while UFO peptides showed potential interaction. This work require further investigation.WP4. To identify LFY interactome and associated PTMsData is currently under analysis.

Progress was slower than expected. However, state of the art technologies applied paved way for follow up experiments. E.g. a combination of chemical crosslinking and MS would be used to confirm LFY and UFO interacting residues. Also, interactome MS data will shed light on the interactors of LFY and in deducing a network of events critical in flower formation. This research sheds light on key mechanisms in flower formation, which will allow modifications on the architecture of plant inflorescences. This has high potential in agriculture through breeding to tackle the social need of food security. So far, our project has caught attention of young generations at primary school through discussions and practicals that allowed them to gain basic understanding on plant growth and flowering. This forms the basics in nurturing love for science to the young generation.

Peptides of UFO and LFY obtained after chemical crosslinking and mass spectrometry.

Yeast-2 hybrid assays showing that LFY and UFO interact.

Pictorial view showing the location of the peptides identified in Table 1.

Western blots to visualize MBP-UFO and HIS-LFY.

Western blots depicting LFY identification and the presence of PTMs in its interactome.